The present invention relates generally to methods and products for activating dendritic cells. In particular, the invention relates to oligonucleotides which have a specific sequence including at least one unmethylated CpG dinucleotide which are useful for activating dendritic cells.
In the 1970s, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., et al., 1971, Proc. Natl. Acad. Sci. USA, 68:1212; Aggarwal, S. K., et al., 1975, Proc. Natl. Acad. Sci. USA, 72:928). In 1985, Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction; binding is saturable, competitive, and leads to DNA endocytosis and degradation into oligonucleotides (Bennett, R. M., et al., J. Clin. Invest., 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., et al. and J. S. Cohen, 1991, Advanced Drug Delivery Reviews, 6:235; Akhtar, et al., 1992, in: xe2x80x9cGene Regulation: Biology of Antisense RNA and DNA,xe2x80x9d R. P. Erickson, Eds, Raven Press, Ltd., New York, p. 133; and Zhao, et al., 1994, Blood, 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.
Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen ConA showed enhanced ODN uptake by T but not B cells (Krieg, A. M., et al., 1991, Antisense Research and Development, 1:161).
Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly(IC) which is a potent inducer of interferon (IFN) production as well as a macrophage activator and inducer of NK activity (Talmadge, J. E., et al., 1985, Cancer Res., 45:1058; Wiltrout, et al., 1985, J. Biol. Resp. Mod., 4:512; Krown, S. E., 1986, Sem. Oncol., 13:207; and Ewel, C. H., et al., 1992, Canc. Res., 52:3005). It appears that this murine NK activation may be due solely to induction of IFN-xcex2 secretion (Ishikawa, R., and C. A. Biron, 1993, J. Immunol., 150:3713). This activation was specific for the ribose sugar since deoxyribose was ineffective. Its potent in vitro anti-tumor activity led to several clinical trials using poly(IC) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) (Talmadge, et al., cited supra; Wiltrout, et al., cited supra; Krown, et al., cited supra, and Ewel, et al., cited supra). Unfortunately, toxic side effects has thus far prevented poly(IC) from becoming a useful therapeutic agent.
Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace xe2x80x9cB cell differentiation factorsxe2x80x9d (Feldbush, T. L., and Z. K. Ballas, 1985, J. Immunol., 134:3204; and Goodman, M. J., 1986, J. Immunol., 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTL (Feldbush, T. L., cited supra), augment murine NK activity (Koo, G.C., et al., 1988, J. Immunol., 140:3249) and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas, 1990, J. Immunol., 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, cited supra). Recently a 5xe2x80x2 triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human xcex3xcex4 T cells (Constant, P., et al., 1994, Science, 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.
Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell, et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., et al., 1990, J. Clin. Invest., 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina, et al. have recently reported that 260-800 bp fragments of poly(bG).(dC) and poly(dG, dC) were mitogenic for B cells (Messina, J. P., et al., 1993, Cell. Immunol., 147:148). Tokunaga, et al. have reported that poly(dg, dc) induces the xcex3-IFN and NK activity (Tokunaga, et al., 1988, Jpn. J Cancer Res., 79:682). Aside from such artificial homopolymer sequences, Pisetsky, et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, et al., 1991, J. Immunol., 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of microbacterial DNA sequences have demonstrated that ODN, which contains certain palindrome sequences can activate NK cells (Yamamoto, et al., 1992, J. Immunol., 148:4072; and Kuramoto, et al., 1992, Jpn. J. Cancer Res., 83:1128).
Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, et al., 1992, J. Exp. Med., 175:597; Branda, R. S., et al., 1993, Biochem. Pharmacol., 45:2037; McIntyre, K., et al., 1993, Antisense Res. Develop., 3:309; and Pisetesky, et al., 1994, Life Sciences, 54:101). These reports do not suggest a common structure motif or sequence element in these ODN that might explain their effects.
Dendritic cells are considered to be the most potent professional antigen-presenting cells (APC) (Guery, J. C., et al., 1995, J. Immunol., 154:536). Dendritic cells capture antigen and present them as peptide fragments to T cells, stimulating T cell dependent immunity. These powerful APCs have been found in skin, blood, dense tissue, and mucosa, and spleen. Several studies have demonstrated that after human dendritic cells which are isolated from peripheral blood are presented peptide antigen they can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro and ex vivo (Engleman, E. G., 1997, Cytotechnology, 25:1). Several clinical trials are currently underway, based on these findings, using ex vivo manipulation of dendritic cells to generate specific anti-tumor dendritic cells for reimplantation. There has been a growing interest in using dendritic cells ex vivo as tumor or infectious disease vaccine adjuvants (Nestle OF, et al., xe2x80x9cVaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cellsxe2x80x9d, Nat Med, 1998; 4: 328-332; Rosenberg S A, et al., xe2x80x9cImmunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanomaxe2x80x9d, Nat Med, 1998; 4:321-327; Hsu F J, et al., xe2x80x9cVaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cellsxe2x80x9d, Nat Med, 1996; 2: 52-58; Tjoa BA, et al., xe2x80x9cEvaluation of phase I/II clinical trials in prostate cancer with dendritic cells and PSMA peptidesxe2x80x9d, Prostate, 1998; 36: 39-44. Numerous animal models demonstrate conclusively that ex vivo generated DC pulsed with protein antigen can be successfully applied for the immunotherapy of cancer and infectious diseases. (Fields R C, et al., xe2x80x9cMurine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivoxe2x80x9d, Proc Natl Acad Sci, USA, 1998; 95: 9482-9487; Okada H, et al., xe2x80x9cBone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasmsxe2x80x9d, Int J Cancer, 1998; 78: 196-201; Su H, et al., xe2x80x9cVaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable Chlamydiaexe2x80x9d, J Exp Med, 1998; 188: 809-818; DeMatos P., et al., xe2x80x9cPulsing of dendritic cells with cell lysates from either B16 melanoma or MCA-106 fibrosarcoma yields equally effective vaccines against B 16 tumors in micexe2x80x9d, J Surg Oncol, 1998; 68: 79-91; Yang S, et al., xe2x80x9cImmunotherapeutic potential of tumor antigen-pulsed and unpulsed dendritic cells generated from murine bone marrowxe2x80x9d, Cell Immunol, 1997; 179: 84-95; Nair S K, et al., xe2x80x9cRegression of tumors in mice vaccinated with professional antigen- presenting cells pulsed with tumor extractsxe2x80x9d, Int J Cancer, 1997; 70: 706-715.
As described in co-pending parent patent application U.S. Ser. No. 08/960,774 the vertebrate immune system has the ability to recognize the presence of bacterial DNA based on the recognition of so-called CpG-motifs, unmethylated cytidine-guanosine dinucleotides within specific patterns of flanking bases. According to these disclosures CpG functions as an adjuvant and is as potent at inducing B-cell and T-cell responses as the complete Freund""s adjuvant, but is preferable since CpG induces a higher Th1 response and is less toxic. Alum, the adjuvant which is used routinely in human vaccination, induces the less favorable Th2 response. Compared to alum, CpG is a more effective adjuvant. The combination of CpG and alum was found to produce a synergistic adjuvant effect.
CpG oligonucleotides also show adjuvant effects towards various immune cells. For instance, CpG enhances the efficacy of monoclonal antibody therapy, thus functioning as an effective immune adjuvant for antigen immunization in a B cell lymphoma model. Cytotoxic T cell responses to protein antigen also are induced by CpG. Furthermore, the presence of immunostimulatory DNA sequences in plasmids was found to be necessary for effective intradermal gene immunization.
It was discovered according to an aspect of the invention that the adjuvant activity of CpG is based on the direct activation of dendritic cells by CpG. Potent immunostimulatory CpG oligonucleotides and control oligonucleotides were found to cause dramatic changes in dendritic cells isolated from peripheral blood by immunomagnetic cell sorting. CpG oligonucleotides provided excellent Dendritic cell survival, differentiation, activation and maturation, and were superior to the combination of GM-CSF and LPS. In fact, the combination of CpG and GM-CSF produced unexpected synergistic effects on the activation of dendritic cells. The invention thus encompasses both CpG oligonucleotides and the combination of CpG oligonucleotides and cytokines such as GM-CSF as well as in vitro, ex vivo, and in vivo methods of activating dendritic cells for various assays and immunotherapeutic strategies.
In one aspect the invention is a method for activating a dendritic cell. The method includes the steps of contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to activate a dendritic cell. In one embodiment the dendritic cell is an isolated dendritic cell.
The isolated nucleic acid is one which contains at least one unmethylated CpG dinucleotide and which is from about 8-80 bases in length. In one embodiment the unmethylated CpG dinucleotide has a formula:
5xe2x80x2N1X1CGX2N23xe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1 is adenine, guanine, or thymine; X2 is cytosine, adenine, or thymine; N is any nucleotide and N1+N2 is from about 0-25 nucleotides. In another embodiment the unmethylated CpG dinucleotide has a formula:
5xe2x80x2N1X1X2CGX3X4N3xe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1X2 is selected from the group consisting of TpT, CpT, TpC, and ApT; X3X4 is selected from the group consisting of GpT,GpA, ApA and ApT; N is any nucleotide and N1+N2 is from about 0-25 nucleotides. In a preferred embodiment N1 and N2 of the nucleic acid do not contain a CCGG quadmer or more than one CCG or CGG trimer. In an illustrative embodiment the isolated nucleic acid is selected from the group consisting of SEQ ID NOS. 20, 24, and 38-46. In another embodiment the isolated nucleic acid is SEQ ID NO.: 84 or 85.
In yet another embodiment the nucleotide of the isolated nucleic acid has a phosphate backbone modification, such as, for example, a phosphorothioate or phosphorodithioate modification. In one embodiment the phosphate backbone modification occurs at the 5xe2x80x2 end of the nucleic acid. Preferably the phosphate backbone modification occurs at the first two intemucleotide linkages of the 5xe2x80x2 end of the nucleic acid. According to another embodiment the phosphate backbone modification occurs at the 3xe2x80x2 end of the nucleic acid. Preferably, the phosphate backbone modification occurs at the last five intemucleotide linkages of the 3xe2x80x2 end of the nucleic acid.
The method for activating the dendritic cell may be performed in vitro, ex vivo, or in vivo. The method in some aspects is a method for cancer immunotherapy, treating an infectious disease, or treating an allergy. When these methods are performed ex vivo they are performed by administering an activated dendritic cell that expresses a specific cancer antigen, microbial antigen or allergen to a subject in need thereof, wherein the activated dendritic cell is prepared by the methods described above. In a preferred embodiment the isolated nucleic acid is administered to a human subject.
In other embodiments the method includes the step of contacting the dendritic cell with a cytokine selected from the group consisting of GM-CSF, IL-4, TNFxcex1, INF-xcex3, IL-6, Flt3 ligand, and IL-3. In yet other embodiments the method includes the step of contacting the dendritic cell with an antigen prior to the isolated nucleic acid.
The invention in another aspect is an isolated antigen-expressing dendritic cell population produced by the process of: exposing an isolated dendritic cell to an antigen; contacting the isolated dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the isolated nucleic acid is from about 8-80 bases in length; and allowing the isolated dendritic cell to process and express the antigen.
The isolated nucleic acid is one which contains at least one unmethylated CpG dinucleotide and which is from about 8-80 bases in length. In one embodiment the unmethylated CpG dinucleotide has a formula:
5xe2x80x2N1X1CGX2N23xe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1 is adenine, guanine, or thymine; X2 is cytosine, adenine, or thymine; N is any nucleotide and N1+N2 is from about 0-25 nucleotides. In another embodiment the unmethylated CpG dinucleotide has a formula:
5xe2x80x2NX1X2CGX3X4N3xe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1X2 is selected from the group consisting of TpT, CpT, TpC, and ApT; X3X4 is selected from the group consisting of GpT,GpA, ApA and ApT; N is any nucleotide and N1+N2 is from about 0-25 nucleotides. In a preferred embodiment N1 and N2 of the nucleic acid do not contain a CCGG quadmer or more than one CCG or CGG trimer. In an illustrative embodiment the isolated nucleic acid is selected from the group consisting of SEQ ID Nos. 20, 24 and 38-46. In another embodiment the isolated nucleic acid is SEQ ID NO.: 84 or 85.
In yet another embodiment the nucleotide of the isolated nucleic acid has a phosphate backbone modification, such as, for example, a phosphorothioate or phosphorodithioate modification. In one embodiment the phosphate backbone modification occurs at the 5xe2x80x2 end of the nucleic acid. Preferably the phosphate backbone modification occurs at the first two intemucleotide linkages of the 5xe2x80x2 end of the nucleic acid. According to another embodiment the phosphate backbone modification occurs at the 3xe2x80x2 end of the nucleic acid. Preferably, the phosphate backbone modification occurs at the last five intemucleotide linkages of the 3xe2x80x2 end of the nucleic acid.
According to another embodiment the isolated antigen-expressing dendritic cell is prepared by contacting the isolated dendritic cell with a cytokine selected from the group consisting of GM-CSF, IL-4, TNFxcex1, INF-xcex3, IL-6, Flt3 ligand, and IL-3.
In yet another embodiment the isolated antigen-expressing dendritic cell is prepared by contacting the isolated dendritic cell with the antigen prior to the isolated nucleic acid.
The invention in another aspect is a composition, including an effective amount for synergistically activating a dendritic cell of an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length; and an effective amount for synergistically activating a dendritic cell of a cytokine selected from the group consisting of GM-CSF, IL-4, TNFxcex1, Flt3 ligand, and IL-3. In an illustrative embodiment the cytokine is GM-CSF. In another embodiment the composition also includes an antigen, such as, for example a cancer antigen, a microbial antigen, or an allergen.
In another aspect the invention is a screening assay for identifying compounds that are effective for preventing dendritic cell maturation. The assay includes the following steps: contacting an immature dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length; exposing the dendritic cell to a putative drug; and detecting the presence or absence of a maturation marker on the dendritic cell, wherein the absence of the maturation marker indicates that the putative drug is an effective compound for preventing dendritic cell maturation. In one illustrative embodiment the maturation marker is CD83.
The invention in another aspect is a method for generating a high yield of dendritic cells. The method includes the following steps administering an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective for activating dendritic cells to a subject; allowing the isolated nucleic acid to activate dendritic cells of the subject; and isolating dendritic cells from the subject.
In another aspect the invention is a method for producing a CD40 expressing dendritic cell. The method includes the following steps: contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to produce a CD40 expressing dendritic cell.
A method for causing maturation of a dendritic cell is provided according to another aspect of the invention. The method includes the step of contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to cause maturation of the dendritic cell.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.